Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

School of Animal Science

First Advisor

Robert A. Godke

Abstract

Goat sperm cells were used as a model to evaluate sperm-mediated gene transfer. Fresh and frozen-thawed goat sperm cells were co-incubated with radio-labelled foreign DNA. After 10 extensive washings, the compacted sperm retained foreign DNA. Confocal microscopy confirmed that the position on the sperm cell responsible for binding of foreign DNA was located in both the anterior and the posterior heads of caprine sperm cells. Furthermore, confocal longitudinal sections of the sperm cells showed that foreign DNA had penetrated into goat sperm nuclei. In this study, 37% of the fresh goat sperm and 33% of the frozen-thawed goat sperm cells were able to bind foreign DNA. In vitro fertilization experiments were conducted with goat sperm cells co-incubated with foreign DNA and the results showed that 3% of the fertilized and cleaved embryos expressed foreign DNA. Electroporation was applied to sperm cells co-incubated with foreign DNA and at various voltages (range of 0 to 1,200 volts/cm). Even though the sperm cells were immobilized by electroporation, all of them were able to bind foreign DNA. A further study showed that the sperm membranes play a very important role in sperm cell binding of foreign DNA. Southern blotting results showed that enzymes in the sperm cells were able to degrade foreign DNA. To further evaluate the potential use of sperm-mediated gene transfer, intracytoplasmic injection of goat oocytes with immotile sperm was evaluated. Results indicated that goat oocytes need exogenous stimuli, such as calcium ionophore A23187 for activation. Overall, 21% of cleaved sperm-injected embryos developed to the blastocyst stage, while sham-injected oocytes only developed to 16- to 32-cell stages. With this goat intracytoplasmic sperm injection methodology, sperm-mediated gene transfer was further studied. Fluorescent in situ hybridization results showed that without electroporation of sperm cells, only 14% of the cleaved embryos had foreign DNA (BC31), while with electroporation, 25% cleaved embryos had foreign DNA (BC31). Furthermore, foreign DNA (BC31) had integrated into goat genomic DNA.

ISBN

9780591458596

Pages

158

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